This proposal aims to understand key innate immune mechanism(s) leading to liver injury following chronic alcohol ingestion. How cellular interactions among innate immune cells in liver, including NKT cells provide both tolerance to gut or metabolic antigens and at the same time immunity against pathogens is poorly understood. NKT cells are comprised of two distinct subsets, type I and type II, and recognize different lipid antigens presented by CD1d molecules. A major subset of type II NKT cells that we identified recognizes a self-glycolipid, sulfatide. In two different models of liver injury we found that sulfatide-mediated activation of type II NKT cells leads to engagement of a novel immune regulatory pathway resulting in anergy induction in type I NKT cells, blockade of the inflammatory cascade, and inhibition of tissue damage. Here we hypothesize that differential activation of NKT cell subsets play opposing roles in liver injury and their interactions with othe innate immune cells, including DCs and myeloid cells, are decisive in orchestrating inflammatory events leading to ALD. Accordingly, we found that during the preclinical phase of ALD following chronic plus binge feeding of Lieber-DeCarli liquid diet in male C57BL/6 mice, type I but not type II NKT cells are partially activated leading to recruitment of inflammatory cells into liver. A central finding we have made is that inactivation of type I NKT cells following administration of sulfatide or all-trans retinoic acid (ATRA) blocks ALD. Furthermore, J?18-/- mice deficient in type I NKT cells are significantly protected from liver injury. Notably, hepatic gene expression profiling revealed type I NKT-dependence of osteopontin (OPN) expression, while sulfatide enhances peroxisome proliferator-activator receptor-? (PPAR?) levels in liver. Cell surface activation markers and the cytokine secretion profile of type I and type II NKT cell subsets during different phases of ALD will be examined using ?GalCer/CD1d- and sulfatide/CD1d-tetramers respectively, and intracytoplasmic staining and flow cytometry. A role for IL-23/IL-17 pathway and adenosine receptor A2aR in activation of type I NKT cells will be examined. Real time PCR and Elisa assays will be used to examine activation of other innate cells in liver during preclinical and clinical phases following ethanol feeding. Adoptive transfer experiments or depletion of conventional cDC or CD11b+Gr-1+ cells, respectively in NKT cell activation and in ALD will be studied. Mechanisms by which administration of ATRA or sulfatide inhibits type I activation leading to blockade of liver damage will be investigated. Sulfatide, ATRA, retinoic acid receptor-? (RAR?) or their combinations along with other inhibitory cytokines will be used to effectively treat ongoing liver disease. Mechanisms of NKT- mediated hepatic expression of OPN and PPAR? genes common in both human and experimental ALD will be examined using siRNA and gene-deficient animals. The proposed studies will provide a better understanding of the key innate immune mechanisms centered on activation of NKT cell subsets following ethanol ingestion and should allow identification of novel immune targets for potential therapeutic intervention in ALD.